In a normal human serum, about 99.97% of thyroxine (T.sub.4) and 99.7% of triiodothyronine (T.sub.3) present, is bound to proteins such as thyroxine-binding globulin (TBG), prealbumin (TBPA) and albumin; the remaining 0.03% of T.sub.4 and 0.3% of T.sub.3 is present as unbound (dialyzable or free) hormones. The serum concentration of thyroid hormones, among other facts such as thyroidal secretion and degradation, is also dependent on serum concentration of TBG; other binding proteins in serum are relatively less important since their binding affinity for thyroid hormones is far less than that of TBG. The concentration of TBG is elevated in conditions such as pregnancy, estrogen treatment or a genetic abnormality; serum total T.sub.4 (and T.sub.3) is increased in these situations. Conversely, serum TBG concentration is decreased during treatment with androgens or due to a genetic deficiency of TBG. This is associated with subnormal concentrations of thyroid hormones. However, in either of the above-mentioned situations of altered TBG concentration, the patient remains eumetabolic and concentrations of free T.sub.4 and (and T.sub.3) and that of total T.sub.4 (and T.sub.3) corrected for TBG abnormality are within the range of normal subjects.
Until a few years ago, serum T.sub.4 concentration was assessed indirectly by measurements of organic (protein bound or butanol extractable) iodine. However, these measurements were frequently erroneous because of iodide contamination in the laboratory or of administration of iodide containing drugs to the patients. In order to overcome these problems, a competitive-protein binding assay (CPBA) for T.sub.4 was introduced by Murphy and Pattee (1). This procedure employs the principal of saturation analysis, and quantitates T.sub.4, relatively specifically, by measurement of displacement of radioactive T.sub.4 from T.sub.4 -binding sites on TBG. It allows measurements of T.sub.4 in a range of 3-20 ug % using a butanol-ethanol extract of serum. In the Murphy-Pattee method, one must first extract thyroxine from the blood serum with alcohol or butanol-ethanol; this introduces error since the extraction procedure itself results in the extraction of varying amounts of thyroxine. The drying of the extract is also time consuming, and must be accomplished before one commences the measurement of T.sub.4.
The Murphy-Pattee procedure thus leaves one with a dried extract containing T.sub.4 as well as much non-thyroxins, such as lipids. To the tubes containing the dried extract, one then adds a 1/32 diluted human serum containing radioactive T.sub.4 (T.sub.4 *). The T.sub.4 in the dried extract competes with T.sub.4 * for binding sites on TBG. After equilibrium is reached one separates the unbound T.sub.4 by means of an ion exchange resin and measures the amount of radioactive counts (of T.sub.4 *) remaining bound to TBG. The quantitation of T.sub.4 is accomplished by reading from a standard curve, prepared simultaneously with known amounts of T.sub.4. FNT (1) Murphy and Pattee J. Lab. Clin. Med. 66.161 July 1965
The disadvantages of the Murphy-Pattee method are that it requires an extraction procedure; it is not sensitive enough and it is cumbersome for processing a large number of samples.
A radioimmunoassay (RIA) has been previously developed and reported, by the applicant herein in conjunction with others, which employs a highly specific antibody to T.sub.4 for its subsequent measurement instead of utilizing TBG from human serum. In this method, a rabbit anti-thyroglobulin antiserum is employed as the T.sub.4 binding protein. Further, in this method, the T.sub.4 must be first extracted as in the Murphy-Pattee method but the method is more sensitive than Murphy-Pattee. This RIA method of applicant herein is reported, in detail, in Journal of Clinical Endrocrinology, 33:865, 1971, incorporated herein by this reference. In general, the precision, reproducibility and practicality of this RIA is comparable to those of competitive protein binding assay (CPBA) using serum TBG as the T.sub.4 binding protein.